The long-term objectives of this application is to understand how B-cells transition through immature and mature stages of differentiation and come to distinguish between foreign and self-antigens and respond to foreign-antigens. Mutations in the Aiolos gene interfere with those processes and provide a novel insights into their mechanism. The goal is to integrate an impressive body of pre-existing research on cell surface events and their signaling cascades with nuclear events that promote cellular responses. Aiolos provides us with a novel and critical entry point to study the progression from cell surface signaling to the nuclear response that regulates an immune reaction. Specific Aim 1 proposes to employ a phenotypic, molecular and cell cycle characterization on the pro-B and pre-B-cell populations in Aiolos deficient mice. These studies will address whether and how Aiolos regulates (in a negative fashion) the specification, proliferative expansion and differentiation of early B-cell precursors. Specific Aim 2 proposes to determine, in a series of genetic and cell biological studies, when Aiolos exerts its effects on B-cell tolerance. During B-cell selection in the bone marrow or during the course of an immune response in the periphery and what are the cellular components involved in this process? Specific Aim 3 probes the molecular underpinnings that cause the B-cell hyperproliferative phenotype in the Aiolos null mice. Cell cycle entry and progression studies in mature activated B-cells which lack Aiolos address the potential molecular defects in this cellular process which may also affect Ig somatic hypermutation and isotype switching. Specific Aim 4 seeks to link cell surface events with changes in the activity of the Aiolos nuclear factor during the course of a B-cell response. How does a normal B-cell modifies the Aiolos complex during the course of its activation and how such temporary changes in the Aiolos complex affect its ability to enter the cell cycle, somatically hypermutate its Ig Ioci and switch to a different isotype are questions addressed under this study. Confocal microscopy and immunoprecipitations as well as a series of biochemical and expression assays are used to study the changes in the Aiolos protein complex during the B-cell cycle and correlate them with the ongoing molecular processes of transcription and DNA replication. Understanding the molecular controls of B-cell differentiation, proliferation and homeostasis are of paramount importance for establishing the molecular underpinnings of immunoregulation and its malfestations i.e. immuno-deficiency, autoimmunity and neoplastic transformation. In the long run, these studies on the role of Aiolos in B-cell differentiation and function, we will have a great impact on our ability to apply molecular intervention to our immune system.